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Introduction. The study of the systemic exposure of a new original drug is an essential part of its preclinical study. 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide is a new selective carbonic anhydrase II inhibitor for the treatment of open-angle glaucoma. Methods for the quantitative determination of this compound and its N-hydroxy- and N-acetyl metabolites in the plasma of laboratory animals have not been previously developed.Aim. Development and validation of a method of quantitative determination of 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide and its metabolites N-hydroxy-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide (M1) and N-acetyl-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide (M2) in rat and rabbit blood plasma by HPLC-MS/MS.Materials and methods. Protein precipitation by methanol was applied for sample preparation. 5-[2-(morpholine-4-carbonyl)-1,3-oxazole-5-yl]-thiophene-2-sulfonamide was used as an internal standard. A 5 % aqueous solution of ascorbic acid was added to the plasma samples at volume ratio 1 : 2 to prevent decomposition of N-hydroxy-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide. The combination of sodium fluoride and potassium oxalate was selected as an anticoagulant. Chromatographic separation was performed on Zorbax Eclipse Plus C18 column (150 × 3.0 mm, 3.5 µm) with Zorbax Eclipse Plus C18 pre-column (12.5 × 2.1 mm, 5.0 µm) using a mobile phase based on a 0.1 % aqueous solution of formic acid and methanol. Mass spectrometric detection was carried out in the MRM mode using electrospray ionization in negative polarity. The method was tested during a pharmacokinetic study of a 1 % ocular suspension of 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide on 6 Wistar rats. Blood samples were collected before administration, as well as 30 min, 1 h, 1 h 30 min, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h, 144 h, 216 h after administration. The non-compartment approach was used for calculation pharmacokinetic parameters.Results and discussion. The developed method has been validated in parameters of selectivity, calibration curve, accuracy and precision, matrix effect, dilution integrity, carry over, reinjection reproducibility, stability. The analytical range of determination of 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide in plasma was 10–4000 ng/ml, M1 – 1.0–400.0 ng/ml, M2 – 0.1–40.0 ng/ml. The selected combination of anticoagulant and stabilizer solution allows storage of plasma samples in freezing chamber for 28 days.Conclusion. The developed method has been fully validated and confirmed its suitability for quantitative determination of 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide and its metabolites in the blood plasma of laboratory animals. The method has been successfully used for pharmacokinetic study of 1 % ocular suspension of the drug.

INTRODUCTION. Systemic exposure studies of a selective carbonic anhydrase II inhibitor, the isoxazole derivative 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide (TFISA), require evaluating its pharmacokinetics in whole blood because the compound can accumulate in erythrocytes. Currently, no bioanalytical procedures have been developed to achieve this.AIM. This study aimed to develop a bioanalytical procedure for the determination of TFISA and its metabolites (N-hydroxy-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide and N-acetyl-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide) in the blood of laboratory animals and compare the pharmacokinetics of TFISA ophthalmic suspension in rats after a single ocular or intraperitoneal administration.MATERIALS AND METHODS. The quantitative determination was performed by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) using rat and rabbit blood samples. The chromatographic separation used a Zorbax Eclipse Plus C18 column (150×3.0 mm, 3.5 µm) and a gradient elution system of 0.1% aqueous formic acid and methanol. The multiple reaction monitoring mass spectrometry mode was used for detection. The pharmacokinetics study was conducted in 2 groups of 6 Wistar rats (3 males and 3 females per group). Group 1 received an instillation of 1% TFISA ophthalmic suspension in each eye at a dose of 3.7 mg/kg. Group 2 received an intraperitoneal injection of the same product at the same dose. Blood samples were collected at baseline and at several intervals after administration.RESULTS. The authors developed a bioanalytical procedure for the determination of TFISA and its metabolites in the blood of laboratory animals (rabbits and rats). This HPLC-MS/MS procedure was fully validated in accordance with the requirements of the EAEU legislation and the ICH M10 guideline. The analytical ranges in blood were 20–20000 for TFISA, 2–2000 for the N-hydroxy metabolite, and 0.1–100.0 ng/mL for the N-acetyl metabolite. The maximum blood levels after ocular instillation (mean±SD) were 8173±1491 for TFISA, 694±271 for the N-hydroxy metabolite, and 6.33±1.51 ng/mL for the N-acetyl metabolite. The half-lives for this route of administration were 58±10 (TFISA), 70±24 (N-hydroxy metabolite), and 14±3 h (N-acetyl metabolite). The bioavailability of TFISA was 90.18%. CONCLUSIONS. The developed and validated bioanalytical procedure for the determination of TFISA and its metabolites in the blood of laboratory animals has been successfully applied to samples of rat whole blood. According to the study of ophthalmic suspension pharmacokinetics, TFISA and its metabolites have long halflives and high bioavailability.

Background. Determining changes in the content of monoamine neurotransmitters and their metabolites in brain structures is a necessary part of studying the pharmacodynamics of antiparkinsonian drugs. A method for the joint determination of norepinephrine, adrenaline, dopamine, serotonin, 5-hydroxyindole-3-acetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid in rat brain tissue has not previously been developed.The aim of the study. To develop and to validate a method for the quantitative determination of norepinephrine, adrenaline, dopamine, serotonin, 5-hydroxyindole3-acetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid in rat brain tissue using high-performance liquid chromatography in combination with tandem mass spectrometry (HPLC-MS/MS).Materials and methods. A method for determining monoamine mediators and their metabolites was developed using the HPLC-MS/MS method. Brain tissue homogenates were prepared using a mechanical hand-operated homogenizer. The effect of various antioxidants on the stability of norepinephrine, adrenaline, dopamine and 3,4-dihydroxyphenylacetic acid in the test samples was studied.Results. Chromatographic separation of sample components was carried out using two Synergi Max RP (20 × 2.0 mm, 2.5 µm) and Synergi Fusion RP 80Å (250 × 4.6 mm, 4 µm) chromatographic columns. Elution was carried out in a gradient mode using a mobile phase based on methanol and a 0.1% solution of formic acid in water. To prepare homogenate batches, the samples were diluted with a solution of internal standards in methanol. A 5% aqueous solution of ascorbic acid was chosen as an antioxidant stabilizer.Conclusion. The developed methodology has been fully validated and meets the requirements of Russian and international guidelines. The chosen stabilization method allows samples of brain homogenates to be stored for 30 days after collection.

The aim of the study was to determine biotransformation products of a new selective carbonic anhydrase II inhibitor – 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide.Materials and methods. The study was conducted on 3 Wistar rats and 3 rabbits of the Soviet Chinchilla breed. The suspension of the drug was administered intraperitoneally to rats at a dosage of 20 mg/kg, to rabbits - at a dosage of 1.6 mg/kg. The animal blood samples were collected before the administration and 1, 2, 4, 24 h after. Urine sampling was also performed in the rats before the administration and in the intervals of 0–4, 4–8, 8–24 h after. The identification of metabolites in blood, urine and plasma was carried out using HPLC-MS/MS. Poroshell 120 C 18 column (50×3.0 mm, 2.7 µm) with a Zorbax Eclipse Plus C18 pre-column (12.5×2.1 mm, 5.0 µm) was used for the chromatographic separation. The assumed metabolites were synthesized, their structure was confirmed by the NMR spectroscopy method and a high-resolution mass spectrometry. The obtained substances were compared with the substances identified in biological fluids by retention time, the main MRM-transitions and mass spectra.Results. The N-hydroxymetabolite was revealed in the analyses of plasma, blood and urine samples which had been formed by the addition of an oxygen atom to the drug molecule. Chromatographic peaks of this compound were identified at the MRM-transitions of 255→159, 255→117, 255→89 m/z at the 7.2nd min of the analysis. The N-oxide of 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide and N-hydroxy-4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide were synthesized; potentially, they could have been obtained during the biotransformation. During the confirmatory HPLC-MS/MS tests based on the coincidence of the retention times, the main MRM transitions and mass spectra, the ratio of the peak areas at the identified metabolite it was established that an N-hydroxy derivative. Chromatographic peaks of the N-oxide detected in the analysis of the model mixtures of the standard substance at the MRM-transitions of 255→175, 255→133, 255→89 m/z at the retention time of 5.43 min, were absent in the animal samples.Conclusion. The studied drug is metabolized to form a single metabolite of N-hydroxy-4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide. This compound was found in freshly collected samples of biological fluids of both animal species. The structure of the metabolite was confirmed by the HPLC-MS/MS-method by comparison with the synthesized standard substance.